Monoclonal antibodies having property of causing apoptosis

ABSTRACT

It is the objective and purpose of the present invention to provide a monoclonal antibody having the property of causing apoptosis on myeloid cells. 
     This invention relates to a monoclonal antibody having the property of causing apoptosis on myeloid cells, and fragments thereof, and furthermore relates to a hybridoma producing the monoclonal antibody. 
     Since the monoclonal antibodies of the present invention are useful as antibodies recognizing and identifying antigens causing apoptosis on myeloid cells specifically and besides have the property of causing apoptosis on myeloid cells, they may be used as medicine useful in the field of remedies for myelocytic leukemia utilizing the property.

TECHNICAL FIELD

The present invention relates to a novel monoclonal antibody having theproperty of causing apoptosis on myeloid cells and being useful asmedicine for myelocytic leukemia, and fragments thereof, andfurthermore, relates to a hybridoma producing the monoclonal antibody.

Since the monoclonal antibodies of the present invention are useful asantibodies recognizing and identifying antigens causing apoptosis onmyeloid cells specifically and besides have the property of causingapoptosis on myeloid cells, they may be used as medicine useful in thefield of remedies for myelocytic leukemia utilizing the property.

BACKGROUND ART

Granulocyte colony-stimulating factors, for example, recombinantgranulocyte colony-stimulating factors (rG-CSF), have been knownprimarily as humoral factors to stimulate the differentiation andproliferation of granulocyte cells, and it has been reported in anexperiment upon mice in vivo that the administration of rG-CSF enhancesthe hematopoiesis of the bone marrow and in addition causes remarkableextramedullary hematopoiesis in the spleen to proliferate hematopoieticstem cells and all hematopoietic precursor cells in the spleen. And ithas been thought as extramedullary hematopoietic mechanism in the spleenthat hematopoiesis occurs due to a splenic hematopoieticmicroenvironment modifications according to the stimulation of rG-CSF toenhance hematopoietic potential.

Hence, the present inventors have noted splenic stromal cellsadministered rG-CSF with a view to clarifying the hematopoieticpotential in the spleen, and established a hematopoietic stromal cellline (CF-1 cells) from the spleen of a mouse administered rG-CSF with aview to attempting the analysis of the enhancement of the hematopoieticpotential by stromal cells with rG-CSF, and examined the potentialeffect on hematopoiesis using the hematopoietic stromal cells, and as aresult, colony-stimulating activities in vitro and potency supportive ofhematopoietic stem cells in vivo have been recognized Blood, 80, 1914(1992)!.

However, though some of splenic stromal cells have been established as acell line (CF-1 cells), and cytological characteristics thereof havebeen examined, no specific antibody recognizing surface antigens thereofhas been prepared so far, and characteristics thereof have been scarcelyknown yet.

Hence, the present inventors have engaged in assiduous studies with aview to developing specific antibodies capable of recognizing splenicstromal cells on the basis of the above information upon splenic stromalcells and the results of the studies, and prepared monoclonal antibodiesusing the splenic stromal cell lines as antigens for immunization, andas a result, novel monoclonal antibodies unreported so far have beenobtained.

And as a result of examining the properties of the obtained monoclonalantibodies, the inventors have found surprisingly that they have theproperty of causing apoptosis on myeloid cells, which has led to thecompletion of the present invention.

DISCLOSURE OF INVENTION

It is the objective and purpose of the present invention to provide anovel monoclonal antibody having the property of causing apoptosis onmyeloid cells and being useful as medicine for myelocytic leukemia, andfragments thereof, and in addition a hybridoma producing the monoclonalantibody.

The monoclonal antibody of the present invention is remarkably useful asan antibody recognizing antigens causing the apoptosis it is also calledself-destruction of cells, phenomenon that a nuclear chromatin DNA isdigested at a nucleosome unit (so-called ladder formation) to result inthe death of cells! of myeloid cells and having a function ofidentifying them or a function of causing apoptosis on myeloid cells.Incidentally, myeloid cells include cells other than lymphoid cells,such as neutrophils, megakaryocytes, myeloblasts, myelocytes, mastcells, macrophages, monocytes and erythroblasts, and the myeloid cellsaccording to the present invention also mean the same as mentionedabove. No monoclonal antibody having the property of causing apoptosison myeloid cells has been known so far, and hence the monoclonalantibodies of the present invention are defined to include allmonoclonal antibodies having the property of causing apoptosis onmyeloid cells.

The monoclonal antibody of the present invention may be preparedbasically as stated below.

Namely, the monoclonal antibody of the present invention may beprepared, for example, by using splenic stromal cells derived from ananimal administered rG-CSF as antigens, immunizing them according to anordinary immunization method, cell-fusing the immunized cells accordingto an ordinary cell fusion method, and cloning the fused cells accordingto an ordinary cloning method.

As a method of preparing the monoclonal antibody of the presentinvention can be preferably exemplified a method comprising using CF-1cells, splenic stromal cells of an animal administered rG-CSFestablished as culture cell line by the present inventors, as theantigen Blood, Vol. 80, 1914 (1992)!, fusing plasma cells (immunocyte)of a mammal immunized with the antigen with myeloma cells of a mammalsuch as a mouse, cloning the obtained fused cells (hybridomas),selecting clones producing antibody according to the present inventionrecognizing the above cell line among them, and culturing them torecover objective antibody. However, the method is only an example, andin this case, for example, not only the above CF-1 cells but also celllines derived from human splenic stromal cells obtained according to thecase of CF-1 cells may be used as the antigens properly to prepareantibodies binding to objective human myeloid cells in the same manneras in the case of the above CF-1 cells.

In the method of preparing such monoclonal antibodies, mammals to beimmunized with the above antigen are not particularly restricted; it ispreferable to make selection taking into account suitability withmyeloma cells to be used in cell fusion, and preferably a mouse, a ratand a hamster are used.

Immunization is performed according to an ordinary method, for example,by administering splenic stromal cells such as the above CF-1 cells intoabdominal cavity of a mammal by injection. More specifically, it ispreferable to administer one diluted with or suspended in a properamount of PBS or isotonic sodium chloride solution to an animal severaltimes every month. It is preferable to use splenic cells removed afterthe final administration of the above cells as immunocytes.

As a myeloma cell of a mammal as the other parent cell fused with theabove immunocytes can be used preferably known various cells includingP3(P3X63Ag8.653) J. Immunol., 123, 1548 (1978)!, p3-U1 Current Topics inMicro-biology and Immunology, 81, 1-7 (1978)!, NS-1 Eur. J. Immunol., 6,511-519 (1976)!, MPC-11 Cell, 8, 405-415 (1976)!, Sp2/0-Ag14 Nature,276, 269-270 (1978)!, FO J. Immunol. Meth., 35, 1-21 (1980)!, S194 J.Exp. Med., 148, 313-323 (1978)! and R210 Nature, 277, 131-133 (1979)!.

The cell fusion of the above immunocyte and a myeloma cell may beperformed basically according to an ordinary method, for example, amethod by Milstein et al. Methods Enzymol., 73, 3-46 (1981)!.

More specifically, the above cell fusion may be performed, for example,in an ordinary nutrition medium in the presence of a fusion-acceleratingagent. As a fusion-accelerating agent, polyethylene glycol (PEG) andSendai virus (HVJ), and furthermore, adjuvants such as dimethylsulfoxide may be added properly if required in order to enhance thefusing effect. Regarding the ratios of immunocytes and myeloma cells,the former is preferably used in an amount 1-10 times that of thelatter. Examples of a medium used in the above cell fusion include aRPMI-1640 medium and a MEM medium suitable for the proliferation of theabove myeloma cell and other mediums ordinarily used for the culture ofthis kind of cell, and in addition, supplementary serum such as fetalbovine serum (FBS) may be used together.

Cell fusion is performed by mixing prescribed amounts of the aboveimmunocytes and myeloma cells in the above medium, adding a PEG solutionpreheated to about 37° C., for example, PEG with an average molecularweight of the order of 1,000-6,000 to the medium, ordinarily, at aconcentration of about 30-60 % (W/V), and mixing them. Subsequently, byrepeating the operations of adding proper mediums one after another,centrifuging the reaction mixture and removing the supernatants can beformed objective hybridomas.

Said hybridomas are selected by culturing in an ordinary selectivemedium, for example, a HAT medium (medium supplemented withhypoxanthine, aminopterin and thymidine). The culture in the HAT mediumis continued for a period sufficient for cells other than objectivehybridomas (non-fused cells) to die out, ordinarily for several days toseveral weeks. Subsequently, the screening and monocloning of thehybridomas producing the objective antibodies are performed according toordinary limiting dilution analysis.

The prepared hybridomas producing the monoclonal antibodies of thepresent invention may be subcultured in an ordinary medium and stored inliquid nitrogen for a long time.

In order to collect the monoclonal antibodies of the present inventionfrom the hybridomas may be employed a method comprising culturing thehybridomas according to an ordinary method, and obtaining them from thesupernatants, or a method comprising administering a hybridoma into aappropriate mammal to proliferate, and obtaining them from its ascite.The former is suitable for obtaining antibodies with a high purity andthe latter is suitable for the mass production of antibodies.

Furthermore, the antibodies obtained according to the above methods maybe purified to a high degree employing an ordinary purification meanssuch as a salting-out technique, gel filtration and affinitychromatography.

The monoclonal antibody of the present invention may be any one so faras it has a specific property to be described specifically in Examplelater, namely, a property of causing apoptosis on myeloid cells, andthose having the property are included in the scope of the presentinvention, irrespective of the kind of antigens; the monoclonal antibodyof the present invention may be used as useful medicine for myelocyticleukemia according to utilizing the property.

Needless to say, the establishment of a specific system for identifyingand recognizing antigens causing apoptosis on myeloid cells according toutilizing the monoclonal antibody of the present invention, or for usingit as medicine for myelocytic leukemia according to utilizing thespecific property thereof, and modification and application thereof areincluded within the scope of the present invention so far as they areput into practice according to an ordinary method obvious to thoseskilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an analysis (a control in the absence of an antibody, CF-1cell) according to immunofluorescence.

FIG. 2 shows an analysis of the binding properties of the GSPST-1antibody to CF-1 cells according to immunofluorescence.

FIG. 3 shows an analysis of the binding properties of the BMAP-1antibody to CF-1 cells according to immunofluorescence.

FIG. 4 shows an analysis (a control in the absence of an antibody, bonemarrow cell) according to immunofluorescence.

FIG. 5 shows an analysis of the binding properties of the GSPST-1antibody to bone marrow cells according to immunofluorescence.

FIG. 6 shows an analysis of the binding properties of the BMAP-1antibody to bone marrow cells according to immunofluorescence.

FIG. 7 shows an analysis (a control in the absence of an antibody,NFS-60) according to immunofluorescence.

FIG. 8 shows the binding properties of the GSPST-1 antibody to NFS-60cells according to immunofluorescence.

FIG. 9 shows an analysis (a control according to rat IgG1, NFS-60)according to immunofluorescence.

FIG. 10 shows the binding properties of the BMAP-1 antibody to NFS-60cells according to immunofluorescence.

FIG. 11 shows an assay for the monoclonal antibody (BMAP-1) to inhibitNFS-60 cell proliferation.

FIG. 12 shows an assay for the monoclonal antibody (GSPST-1) to inhibitthe bone marrow transplantation.

FIG. 13 shows an assay for the monoclonal antibody (BMAP-1) to inhibitthe bone marrow transplantation.

FIG. 14 is an explanatory view microphotograph (stained with H. E.) ofbone marrow samples (×400)! showing dead bone marrow cells (2) on 6 daysafter the administration of the monoclonal antibody BMAP-1 of thepresent invention, and the control (1) in the absence of the antibody.

FIG. 15 is an explanatory view (migration-photo according toelectrophoretic chromatography) showing the ladder formation of the DNAof bone marrow cells observed when the monoclonal antibody BMAP-1 of thepresent invention was administered.

FIG. 16 shows a cytotoxicity assay using L929 cells by TNF α

FIG. 17 shows a cytotoxicity assay by the monoclonal antibody (BMAP-1).

FIG. 18 shows an analysis (a control according to rat IgG2a, BWV1)according to immunofluorescence.

FIG. 19 shows the binding properties of the anti-mouse MHC class Iantibody to BWV1 cells according to immunofluorescence.

FIG. 20 shows an analysis (a control according to rat IgG1, BWV1)according to immunofluorescence.

FIG. 21 shows the binding properties of the BMAP-1 antibody to BWV1cells according to immunofluorescence.

EXPLANATION OF SYMBOLS

a: DNA of the thymus of a mouse administered BMAP-1 (24 hours)

b: DNA of the bone marrow of a mouse administered BMAP-1 (24 hours)

c: DNA of the bone marrow of a mouse administered BMAP-1 (8 hours)

d: DNA of the bone marrow of a mouse administered BMAP-1 (4 hours)

e: DNA of the bone marrow of a non-treated mouse (bone marrow cells)

f: Molecular weight marker

Next, the present invention will be described further in detailaccording to Reference Example and Example, but the present invention isnot restricted to the Example.

Reference Example Establishment of Splenic Stromal Cells and TheirCharacteristics Thereof

1) Establishment of Splenic Stromal Cells

A splenic stromal cell line was established from the primary culturmousthe splenic cells of a C57BL/6J mouse administered rG-CSF 100 μg/kg for5 days. Namely, this spleen was removed after the administration ofrG-CSF under germ-free conditions, cultured in a 25-cm² plastic flask(Corning Co.) for 6 weeks and in an Isocove's modified Dulbecco's medium(IMDM) (Boehringer-Mannheim Co.) with 10% heat-inactivated fetal bovineserum (FBS) (Sanko Junyaku, Tokyo), 100 U/ml penicillin and 100 μg/mlstreptomycin in an incubator under the condition of 37° C. and 5% CO₂,and the medium was exchanged for a fresh growth medium twice a week.

In the confluent culture, the adherent cell populations (stromal cells)were harvested from the flask by using 0.05% trypsin plus 0.02% EDTA(Sigma Chemical Co.) in Ca-, Mg-free PBS, and were transferred into newflasks. These passages were repeated approximately once or twice a week.In the early passages (1st through 10th passages), the split ratio ofthe cells was 1/4 to 1/8, and subsequently the ratio was 1/16 to 1/32.The stromal cells became homogeneous and fibroblastoid afterapproximately the 10th passage. At the 20th passage, these stromal cellswere harvested as described above and forwarded to cell cloning by usinga limiting dilution technique; cell cloning was repeated twice toestablish a stromal cell line (CF-1 cell line).

Subsequently, these cells were maintained in 5 ml of IMDM supplementedwith 10% heat-inactivated FBS in a 25-cm² flask (Corning Co.), andsubcultured once every 5 days at the split ratio of 1/32. Splenicstromal cell lines can be established from other animals than mouse; forexample, human splenic stromal cell lines can be established using thesame method as described above by transforming the cells with an SV-40adenovirus vector J. Cell. Physiol., 148, 245 (1991)!.

2) Characteristics of CF-1 Cells

CF-1 cells established as a cell line as described above were examinedfor alkaline phosphatase, acid phosphatase, β-glucuronidase, α-naphthylacetate esterase and oil red O using standard cytochemical techniques.CF-1 cells were also characterized by immunoenzymatic histochemistryusing the following monoclonal and polyclonal antibodies: macI (SeroTec.); factor VIII-related antigen (Dakopatts); and collagen type I,collagen type III and fibronectin (Chemicon International Inc.).Phagocytosis was tested by latex bead uptake (particle diameter: 1.09μm; Sigma), and the potency of CF-1 cells to convert to adipocytes wastested by exposure to 10⁻⁶ mol/l hydrocortisone phosphate (Sigma) in a25-cm² flask for 4 weeks after the confluent culture.

As a result, the CF-1 cells were negative for alkaline phosphatase,factor VIII-related antigen, mac I and phagocytosis, whereas they werepositive for collagen type I, collagen type III and fibronectin. CF-1cells were not converted to adipocytes during 4 weeks in a confluentculture with 10⁻⁶ mol/l hydrocortisone, although CF-1 cells had onlytraces of lipid. From these data, CF-1 cells do not have thecharacteristics of preadipocytes, macrophages and endothelial cells, andtherefore it has become obvious that they are derived from stromal cellsdifferent from them.

3) Maintenance of Hematopoietic Stem Cells by CF-1 Cells

To examine whether hematopoietic stem cells are maintained by CF-1 cellsor not, CFU-S assays (assays of spleen colonies-forming cells) wereperformed by the technique of Till and McCulloch. Ten mice per groupwere irradiated with 900 cGy (MBR-1520R; Hitachi, Tokyo) and injectedintravenously with bone marrow mononuclear cells (BM cells) (1.0×10⁵/head, 5.0×10⁴ /head, or 2.5×10⁴ /head) and CF-1 cells (1.0×10⁵ /head),and colonies in the spleen were counted on the 12th day as CFU-S clones(spleen colonies).

As a result, when bone marrow mononuclear cells (BM cells) and CF-1cells were transplanted into irradiated mice, the number of spleencolonies of every group of BM cells increased significantly (between1.4-1.8 times) as compared to the mice without CF-1 cells, and, on the12th day after the transplantation, the survival ratios of the micetransplanted with BM cells and CF-1 cells were higher than those withonly BM cells, showing a low death rate; hence it has become apparentthat hematopoietic stem cells are maintained by CF-1 cells.

BEST MODE FOR CARRYING OUT THE INVENTION

The embodiment of the invention will be described in detail hereinafter.

EXAMPLE Establishment of Monoclonal Antibodies

1) Antigens and Immunization

Immunization was performed by using CF-1 cells obtained in the aboveReference Example as antigens. The cells were cultured in an incubatorunder the condition of 5% CO₂ and 37° C., using an Isocove's modifiedDulbecco's medium (IMDM) (Boehringer-Mannheim Co.) supplemented with 10%fetal bovine serum (FBS; Sanko Junyaku) as a medium.

The cells were treated with 1 mM EDTA/PBS, and removed from a cultureflask according to pipetting. The cells were suspended into 1 mMEDTA/PBS at the cell number of about 1×10⁷ /ml, and administered to aWistar Imamich rat (7-week-old, female; Animal Breeding ResearchLaboratory). One ml of cells of about 1×10⁷ /ml were injected into theabdominal cavity of the rat at the initial immunization, and 1 ml ofcells of about 1×10⁷ /ml were administered additionally one month later.Further, 1 ml of cells of about 1×10⁷ /ml were administered additionallyseveral times at an interval of a month, and after the reactivitybetween the immunized rat antibody and CF-1 cells was recognized, 1 mlof cells of 1×10⁸ /ml were administered as the final immunization. Threedays after the final immunization, the rat was killed to remove spleen.

2) Cell Fusion

After the spleen removed from the rat was minced, splenic cells isolatedwere centrifuged, suspended in an IMDM medium (Boehringer-Mannheim Co.),and washed intensively. On the other hand, the cells obtained bycultured mouse myeloma cell line Sp2/0-Ag14 Nature, 276, 269-270 (1978)!in an IMDM (Boehringer-Mannheim Co.) supplemented with 10% fetal bovineserum (FBS; Sanko Junyaku) were washed in the above IMDM medium in thesame manner, and 1×10⁸ thereof and 2×10⁸ of the above splenic cells wereput into a centrifuge tube and mixed to perform cell fusion bypolyethylene glycol 4000 (Nakarai Kagaku) according to an ordinaryprocedure Clin. Exp. Immunol., 42 , 458-462 (1980)!.

Subsequently, the obtained fused cells were dispensed into a 96-wellplate with an IMDM medium supplemented with 20% FBS, and cultured in anincubator under the condition of 37° C. and 5% CO₂. They were replacedinto a HAT selective medium gradually from the following day, andcontinued to be cultured.

After the start of the culture, the supernatants were replaced into anew HAT medium twice a week to continue the culture and maintain theproliferation.

Next, the obtained fused cells were cloned according to an ordinaryprocedure using limiting dilution analysis. Namely, only clones havingstrong binding properties to antigens were cloned according to anordinary procedure employing limiting dilution analysis by examiningbinding properties thereof to the antigens, utilizing antibodies in thesupernatants of the above fused cells.

3) Screening

The screening of fused cells (hybridomas) was performed according toindirect fluorescent antibody technique using flow cytometry.

The screening of clones producing objective antibodies was performedusing CF-1 cells as target cells. Namely, cells suspended in a reactionbuffer (PBS suppplemented with 2% FBS and 0.02% NaN₃) were centrifugedand recovered as pellets, then suspended in 100 μof the hybridomaculture supernatants (about 1×10⁶ /100 μl) and reacted at 4° C. for 1hour. After they were washed with the above buffer once, anFITC-labelled goat anti-rat IgG (FC) antibody (Chemicon) was addedthereto and incubated for 1 hour. After they were washed once, they wereanalyzed according to flow cytometry (FACScan, Becton Dickinson).

4) Purification of Antibodies

The fused cells screened in the manner of the above 3) were culturedaccording to an ordinary procedure, and antibodies produced in thesupernatants were separated according to an ordinary procedure, andpurified.

Namely, hybridomas were recovered from wells with high antibody titersto the antigens, spread in a tissue culture plastic dish (Corning Co.),cultured under the condition of 5% CO₂ and 37° C., proliferated, andpurified according to an ordinary procedure to obtain monoclonalantibodies GSPST-1 and BMAP-1.

Regarding GSPST-1, obtained cells were injected into the abdominalcavity of a BALB/cAJc1-nu nude mouse (8-week-old, male, Nippon Kurea).Produced ascite was recovered after 10-14 days, salted out with 33%ammonium sulfate, and dialyzed with PBS. Regarding the BMAP-1 antibody,it was cultured in a large scale in an Iscove's modified MEM mediumsupplemented with 10% FBS, and the supernatants were concentrated,salted out with 33% ammonium sulfate, dialyzed with PBS, purified againby means of a protein A column kit (Amersham), and dialyzed with PBS.Incidentally, in the above Example was described the case in which theCF-1 cells were used as antigens for immunization; however, it ispossible to establish a monoclonal antibody in the same manner also incase of using other stromal cells having potency supportive ofhematopoietic stem cells, and the present invention is not restricted tothe above monoclonal antibodies but includes all monoclonal antibodieshaving the same characteristics and all hybridomas producing themonoclonal antibodies.

A hybridoma producing the monoclonal antibody BMAP1 of the presentinvention is a novel fused cell prepared from a Wistar Imamich ratsplenic cell and a mouse myeloma cell line SP2/0-Ag14 as parent cells,and was deposited on 9 Aug., 1993, under the name of BMAP-1 (rat mousehybridoma) with the accession number of FERM BP-4382, at NationalInstitute of Bioscience and Human Technology, Agency of IndustrialScience and Technology in Japan address: 1-3, Higashi 1-chome,Tsukuba-shi, Ibaraki 305, Japan!, international depositary authorityaccording to Budapest Treaty on the international recognition of thedeposit of microorganisms for the purpose of patent procedures.

5) Properties of Antibodies

(i) Reactivity of Antibodies

(Reactivity to CF-1 Cells)

The results of examining the reactivity of the obtained monoclonalantibodies GSPST-1 and BMAP-1 to CF-1 cells according toimmunofluorescence analysis are shown in FIG. 1 through FIG. 3. Here,FIG. 1 shows the results of analysis of the control in the absence of anantibody, FIG. 2 the results of analysis of the binding properties ofGSPST-1 to CF-1 cells, and FIG. 3 the results of analysis of the bindingproperties of BMAP-1 to CF-1 cells. In the drawings, vertical axes showrelative number of cells and transverse axes fluorescence intensity.

As is apparent from FIG. 1 through FIG. 3, it has been revealed thatmonoclonal antibodies GSPST-1 and BMAP-1 have properties binding to CF-1cells and recognize surface antigens of CF-1 cells.

(Reactivity to Bone Marrow Cells)

Next, the results of analysis of the reactivity of GSPST-1 and BMAP-1 tonormal bone marrow cells according to flow cytometry (FACScan, BectonDickinson) are shown in FIG. 4 through FIG. 6. Here, FIG. 4 shows theresults of analysis of the control in the absence of an antibody, FIG. 5the results of analysis of the binding properties of GSPST-1 to bonemarrow cells, and FIG. 6 the results of analysis of the bindingproperties of BMAP-1 to bone marrow cells. In the drawings, verticalaxes show relative number of cells and transverse axes fluorescenceintensity.

As is shown in FIG. 4 through FIG. 6, it has been revealed that GSPST-1has not a property binding to bone marrow cells at all, and that BMAP-1has a property binding to all bone marrow cells.

(Reactivity to Myelocytic Leukemic Cell Line (NFS-60))

The results of analysis of the the reactivity of GSPST-1 and BMAP-1 toNFS-60 cells Proc. Natl. Acad. Sci. U.S.A., 82, 6687-6691 (1985)!according to flow cytometry (FACScan, Becton Dickinson) are shown inFIG. 7 through FIG. 10. Here, FIG. 7 shows the results of analysis ofthe control in the absence of an antibody, FIG. 8 shows the results ofanalysis of the binding properties of GSPST-1 to NFS-60 cells, FIG. 9shows the results of analysis of the control using rat IgG1 on themarket (Zymed) and FIG. 10 shows the results of analysis of the bindingproperties of BMAP-1 to NFS-60 cells. In the drawings, vertical axesshow relative numbers of cells and transverse axes fluorescenceintensity.

As is shown in FIG. 7 through FIG. 10, it has been revealed that GSPST-1does not react with NFS-60 cells, and that BMAP-1 has a property bindingto NFS-60 cells.

(Assay for BMAP-1 to Inhibit Proliferation of NFS-60 Cells)

The results of examining the action of BMAP-1 to NFS-60 cells in thepresence of G-CSF 100 ng/ml and cyclohexyimide 10⁻⁹ M according to theMTT assay method are shown in FIG. 11. Using culture plates with 96wells, 10 μl/well of BMAP-1 solution were added at concentrations of 0,10, 100 ng/ml, and 1, 10, 100 μg/ml to 4×10³ /well/100 μl of NFS-60cells, and two days after the numbers of living cells were measuredaccording to the MTT method. It has been revealed as shown in FIG. 11that the proliferation of NFS-60 cells is inhibited remarkably byBMAP-1.

(ii) Typing of Antibodies

Next, as a result of typing the subclass of IgG of the obtainedmonoclonal antibodies using a rat Mono Ab-ID-Sp kit (Zymed) and abiotin-labelled mouse anti-rat IgG1 antibody (Zymed)!, it has becomeapparent that GSPST-1 is IgG2a, and that BMAP-1 is IgG1.

(iii) Potency Inhibiting Bone Marrow Transplantation

Next, a test upon the inhibition of bone marrow transplantation wasperformed using these antibodies to examine characteristics thereof. Theresults are shown in FIG. 12 and FIG. 13. As is shown in FIG. 12 andFIG. 13, while BMAP-1 has the effect inhibiting the bone marrowtransplantation, the effect has not been found in GSPST-1. Namely, theabove results were obtained by administering 1.0×10⁵ /head of bonemarrow cells and monoclonal antibodies to C57BL/6J mice, irradiated at afatal dose of radiation (900 cGy), through the veins of tails, andcounting the number of spleen colonies. Incidentally, "Non-treated" inFIG. 13 shows the case with no administration of bone marrow cells.

As is shown in FIG. 13, it has been confirmed that it is because BMAP-1reacts with bone marrow cells to cause apoptosis that the monoclonalantibody inhibits transplantation completely in the test upon theinhibition of bone marrow transplantation. Namely, when a hybridomaproducing BMAP-1 was administered into the abdominal cavity of a nudemouse, it died at the time when its ascite was stored in a small amount.In addition it has been revealed that all bone marrow cells died outaccording to the intravenous administration of 50 μg/head BMAP-1 to anormal C57BL/6J mouse, and in FIG. 14 is shown a microphotograph backingup the fact that bone marrow cells on 6 days after the intravenousadministration of BMAP-1 died out. As is apparent from themicrophotograph, it has been observed that not only lymphoid cells butalso neutrophils, megakaryocytes, myeloblasts, myelocytes, mast cells,macrophages, monocytes and erythroblasts (so-called myeloid cells) diedout. In addition, as a result of investigating the DNAs of the bonemarrow cells of a mouse administered 30 μg/head BMAP-1, apparentlyladder formation has been observed as is shown in FIG. 15, and it hasbeen revealed that the above reaction of BMAP-1 to bone marrow cells isdue to apoptosis.

The Fc region of the IgG of the BMAP-1 antibody was digested with pepsin(Sigma) and purified by means of a GPC column as F(ab')2, and 33.5μg/head (corresponding to 50 μg/head of the whole IgG) were administeredto a C57BL/6J mouse intravenously; as a result of it, it was observedthat bone marrow cells died out in the bone marrow. It has becomeapparent according to the above fact that neither antibody-dependentcell cytotoxicity nor complement-dependent cell cytotoxicityparticipates in the cell death of bone marrow cells by BMAP-1.

As an antigen causing apoptosis has been reported the Fas antigen ofcell surface protein; regarding the Fas antigen, the expressions ofmRNAs of it are recognized in the thymus, heart, liver, lungs and ovary,but few mRNAs of it are detected in the bone marrow J. Immunol., 148,1274-1279 (1992)!, and hence it is apparent that antigens recognized byBMAP-1 are different from the conventionally known Fas antigen.

Furthermore, in order to make it clear whether an antigen recognized byBMAP-1 would be a TNF receptor or not, the function of BMAP-1 wasinvestigated using L-929 cells reacting with TNF to cause cell death.The final concentrations of a mouse TNF α (Genzyme) were 0, 1, 10, 100pg/ml, 1, 10, 100 ng/ml, and 1 μg/ml, and those of BMAP-1 were 0, 10,100 pg/ml, 1, 10, 100 ng/ml, and 1, 10 μg/ml, and the numbers of livingcells of L-929 cells were measured according to the MTT method on thesecond day after the addition of the TNFα and BMAP-1. As a result of it,as shown in FIG. 16, FIG. 17, while L-929 cells were reduced by TNF αremarkably, BMAP-1 had no effect upon L-929 cells. Hence, it has becomeapparent that an antigen recognized by BMAP-1 is not a TNF receptor.

The results of investigating whether antigens recognized by BMAP-1 wouldbe MHC class I antigens or not according to flow cytometry (FACScan,Becton Dickinson) are shown in FIG. 18 through FIG. 21. Here, FIG. 18shows the results of analysis of the control using rat IgG1 (Zymed),FIG. 19 shows the results of analysis of the binding properties of theanti-mouse MHC class I antibody (rat IgG2a, BMA) to BWV1 cells (mouselymphoma derived from BW5147 cells), FIG. 20 shows the results ofanalysis of the control using rat IgG1 (Zymed) and FIG. 21 shows theresults of analysis of the binding properties of BMAP-1 to BWV1 cells.In the drawings, vertical axes show relative numbers of cells andtransverse axes fluorescence intensity. As a result, BMAP-1 did notrecognize BWV1 cells but the MHC class I antibody reacted with BWV1cells.

As described above, it has been confirmed experimentally that BMAP-1 hasthe function of causing apoptosis on myeloid cells; according to thepresent inventors knowledge, no monoclonal antibody having the propertyof causing apoptosis on myeloid cells has been reported so far asdescribed above, and hence monoclonal antibodies having such a functionare novel ones found by the present inventors. And, since it is thoughtthat the monoclonal antibodies of the present invention represented byBMAP-1 can cause death of myelocytic leukemic cells considered to behigh in the expression of antigens thereof by utilizing the function ofthe apoptosis of the monoclonal antibody on bone marrow cells, themonoclonal antibody of the present invention having the property ofcausing apoptosis on myeloid cells is useful as medicine for myelocyticleukemia.

The monoclonal antibodies of the present invention have been describedspecifically according to the Example as above; as the monoclonalantibodies having the property of causing apoptosis on myeloid cellsaccording to the present invention may be exemplified those mentioned asspecific examples above, but they are not always restricted to them butinclude all monoclonal antibodies having the same characteristic andfunction prepared in the same manner, irrespective of the kind ofantigens.

Industrial Applicability

Since the monoclonal antibodies of the present invention are useful asantibodies recognizing and identifying antigens causing apoptosis onmyeloid cells specifically and besides have the property of causingapoptosis on myeloid cells, they may be used as medicine useful in thefield of remedies for myelocytic leukemia utilizing the property.

What is claimed is:
 1. A monoclonal antibody that recognizes an antigenexpressed by the splenic stromal cells of an animal administered rG-CSF,said monoclonal antibody having the property of causing apoptosis onbone marrow cells.
 2. F(ab)₂ fragments of a monoclonal antibody thatrecognizes an antigen expressed by the splenic stromal cells of ananimal administered rG-CSF, said monoclonal antibody having the propertyof causing apoptosis on bone marrow cells.
 3. A hybridoma producing amonoclonal antibody that recognizes an antigen expressed by the splenicstromal cells of an animal administered rG-CSF, said monoclonal antibodyhaving the property of causing apoptosis on bone marrow cells.
 4. Themonoclonal antibody of claim 1 prepared by using human splenic stromalcells as antigen.
 5. The monoclonal antibody of claim 1 prepared byusing human splenic stromal cells transformed with SV-40 adenovirusvector as antigen.
 6. A pharmaceutical composition comprising themonoclonal antibody of claim 4 and a pharmaceutically acceptablecarrier.
 7. The monoclonal antibody of claim 1, which is BMAP-1 producedby hybridoma FERM BP-4382.
 8. The hybridoma of claim 3, which is FERMBP-4382.